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Mechanism of Activation of AMPK/P53-CyPD Signalling Pathway in Inhibiting Osteoblast Apoptosis Induced by Dexamethasone
Z. GUO, L. ZHU, C. YIN, Y. LIU, J. FANG AND Y. ZHEN* Department of Orthopaedics, Children’s Hospital of Soochow University, Suzhou Industrial Park, No.92 Zhongnan Street, Suzhou, Jiangsu Province, China
Guo et al: Mechanism of Activation of AMPK/P53-CyPD Signalling Pathway
The objective of this study was to investigate the role of AMPK/P53-CyPD signalling pathway in osteoblast apoptosis induced by dexamethasone and to confirm that the activation of adenosine monophosphate- activated protein kinase signalling pathway can delay the progression of osteonecrosis of the femoral head and promote the regeneration of osteoblasts. In this study, 24 C57BL/6J 8-week old mice were selected as research subjects, which were randomly divided into the model group, the blank group and the intervention group, with 8 mice in each group. The model group was given subcutaneous injection of dexamethasone, the blank group was given subcutaneous injection of the same dose of normal saline, and the intervention group was given intraperitoneal injection of cyclophilin D (CyPD) inhibitor and P53 inhibitor on the basis of the model group. Eight weeks after the experiment, all mice were sacrificed, femoral head samples were taken for nuclear magnetic resonance and micro CT scanning, and the femoral head tissue samples were made for hematoxylin and eosin staining to analyse the effect of inhibiting the AMPK/P53-CyPD signalling pathway on the femoral head structure in mice. Then, the AMPK α-expressing osteoblast cell line was constructed by exogenously adding the AMPK activator acadesine to the femoral head tissue of the model group. The cell survival cycle and apoptosis of osteoblasts were observed. The results showed that firstly, the morphology of the femoral head in the model group changed, the bone trabecula became thinner, the continuity was destroyed and the subchondral bone was partially necrotic. The morphology of the femoral head in the intervention group was significantly changed, the bone density decreased and the bone marrow cavity increased significantly and the structure of the normal femoral head was lost. The number of bone cells in the lacuna was significantly decreased. Secondly, after the exogenous addition of acadesine, the AMPK signalling pathway was activated, the apoptosis was gradually improved and the formation of osteoblasts was significantly increased. It could be concluded that the AMPK/P53-CyPD signalling pathway plays an important role in osteoblast apoptosis induced by dexamethasone, which can inhibit osteoblast apoptosis, delay the process of femoral head osteonecrosis, and promote the recovery of femoral head osteonecrosis.
150 Indian Journal of Pharmaceutical Sciences Special Issue 1, 2019 www.ijpsonline.com Key words: AMPK signalling pathway, mitochondrial P53-CyPD, femoral head osteonecrosis, dexamethasone, osteoblast
Osteonecrosis of the femoral head (ONFH) is a common in mice, and analysed the effect of inhibiting AMPK/ orthopaedic disease caused by a variety of causes of P53-CyPD signalling pathway on apoptosis of bone apoptosis of bone cells and abnormal metabolism of cells; and then the mouse AMPK with high α expression fat cells, resulting in partial or complete ischemic osteoblast cell line was constructed by exogenously necrosis of the femoral head[1]. ONFH usually causes adding AMPK activator acadesine (5-aminoimidazole- changes in bone morphology, which causes femoral 4-carboxamide riboside, AICAR) and the effect of head collapse and osteoarthritis. Therefore, femoral activating AMPK/P53-CyPD signalling pathway on head osteonecrosis is also the main cause of hip joint osteoblast apoptosis induced by dexamethasone was damage[2]. The pathogenesis of ONFH is still unclear. analysed. Trauma, alcoholism, and hormones may cause ONFH. A total of 24 healthy 8 w old C57BL/6J mice of SPF The femoral head osteonecrosis caused by hormones is grade were selected, weighing 20.8±4.3 g, and housed the most common non-traumatic cause, which is more under standard laboratory conditions, the temperature common in young people. was controlled at 22~24°, and the humidity was Application of large doses of hormonal drugs in controlled at 30~40 %. All mice were randomly divided the short term, such as intra-articular injection of into 3 groups, the model group, the blank group and glucocorticoids can cause bone cell apoptosis due the intervention group, with 8 mice in each group. to a series of pathological changes, leading to the The model group received subcutaneous injection of occurrence of ONFH[3]. In the early stage of the 20 mg/kg dexamethasone was, once a day for 8 w. The disease, the lesions mostly occur in the subchondral blank group received subcutaneous injection of same bone. As the disease progresses further, the cartilage volume of physiological saline once a day for 8 w. The collapses in the load-bearing area of the femoral head intervention group was given dexamethasone+CyPD occurs, and finally the hip osteoarthritis. In the 1930s, inhibitor (cyclosporine A, CsA)+P53 inhibitor (PTFα). studies reported that high-dose and continuous use of CsA was injected intraperitoneally at 10 mg/kg, and hormonal drugs may have an effect on bone tissue[4]. PTFα was intraperitoneally injected at 2 mg/kg, once Since then, more and more cases have suggested that a day for 8 w. All mice were given intramuscular there is a certain relationship between hormones and injection of gentamicin sulphate to prevent infection ONFH. However, due to individual differences and the during the experiment, 50 000 U/time, once a week. different courses of treatment and dosage of different After successful model construction, all mice were hormones, the degree of osteonecrosis varies. fed with standard mouse feed, and given free access to drink water. The position of the drinker was raised, AMPK is an adenylate-activated protein kinase, and allowing the mice to be in a standing position while the catabolic pathway also occurs when the AMPK drinking water, increasing the load on the femoral head signalling pathway is activated. Studies have found that to promote the establishment of a ONFH model. The by activating the AMPK signalling pathway, mouse mental state, diet, weight, activity and urination of the embryonic osteoblastic precursor (MC3T3-E1) cells mice were closely observed during modelling. can be induced to differentiate, thereby stimulating the proliferation and differentiation of osteoblasts[5]. Eight weeks after the experiment, all mice were Therefore, regulation of the AMPK signaling pathway sacrificed by cervical dislocation. One side of the can affect the differentiation of osteoblasts. In addition, femoral head was taken out and immersed in 10 ml of P53 gene also has certain effects on apoptosis, a 10 % formalin in a centrifuge tube, and stored in a regulation and metabolism in cells, and P53 protein can refrigerator. Micro CT scan (80 kV; 500 μA; resolution play a crucial role in regulating the differentiation and 10 μm) was performed on the femoral head samples development of osteoblasts through various signalling of mice to obtain continuous scan images. After the pathways[6]. Based on the above theory, this study scan, the bone trabecula and bone marrow cavity, constructed a model of dexamethasone-induced ONFH the bone volume and total volume (BV/TV) scores,
*Address for correspondence E-mail: [email protected] Special Issue 1, 2019 Indian Journal of Pharmaceutical Sciences 151 www.ijpsonline.com thickness and number of bone trabecula, bone area one-way analysis of variance; if p<0.05, the variance to bone volume ratio (BS/BV) and other results were is not uniform, then the one-way analysis of variance quantitatively analysed by computer software. is corrected by Welch; the multivariate analysis is performed by LSD method for comparison between Preparation of specimens of ONFH, firstly, the mouse two pairs, p<0.05 is considered as statistically different. femoral head after micro CT scanning was placed in a centrifuge tube for decalcification, which was In general, the mouse model of early ONFH is considered to be successful when a fine needle can evaluated by imaging and pathological examination. penetrate the bone tissue without resistance. Secondly, Imaging examinations include X-ray, nuclear magnetic the femoral head specimens were dehydrated in ethanol resonance and micro CT and so on. However, X-rays at a concentration gradient of 70, 85, 90 %, and finally have a poor effect on displaying early changes in the treated with 100 % ethanol. Thirdly, xylene was used femoral head of mice, and have limited diagnostic to hyalinize the femoral head sample. Fourthly, the value for early ischemic necrosis of femoral head, transparent specimen was immersed in the paraffin so magnetic resonance imaging and micro CT were liquid. Fifthly, after paraffin embedding, the femoral adopted as the means of modelling evaluation in head tissue of the mouse was sectioned along the this study, which can help clearly observe the bone coronal plane of the femoral neck at a thickness of trabecula and surrounding structures. 4 μm, and separated after warm water bath. Sixthly, By observing the general condition of the mice, it was the section was placed at the center of the glass slide found that in the modelling process of this experiment, and then placed in the oven for bake. After that, it was the gait of the blank group was normal and the stored at room temperature. comprehensive condition was good; while in the model Hematoxylin and eosin (HE) staining of rat femoral group and the intervention group, hair loss occurred head tissue was performed after dewaxing. The above between 4 w and 5 w, the body weight was slightly sample was sliced and baked, then put into xylene and reduced, and the activity was reduced; at 8 w, the hair dewaxed for about 15 min. Secondly, it were placed in loss of the model group and the intervention group was 100, 95, 85 % alcohol in sequence and dehydrated for aggravated, the food intake was reduced, and the mice 3 min. Thirdly, hematoxylin was used for staining for in the model group showed mild lameness, and the mice 3~5 min, and then the section was washed with running in the intervention group were severely paralyzed, and water. Fourthly, the section was differentiated in 1 % most of the mice were unable to walk. Micro CT is hydrochloric acid alcohol and put into the warm water an important method for the early diagnosis of femoral returned to blue and then counterstained with ponceaux head osteonecrosis under the action of hormones. It acid fuchsin for 3 min. Fifthly, after dehydration in has high sensitivity and accuracy, so it was used in this alcohol, it was soaked in xylene, sealed with neutral study as an evaluation method for the establishment gum and dried. of a model of femoral head osteonecrosis in mice. The general condition of each group of mice, including Figs. 1 and 2 show the results of magnetic resonance body weight, activity, diet was observed during the and micro CT scan of the femoral head after 8 w of experiment. The results of nuclear magnetic resonance experiment in all 3 groups of mice. and micro CT scan of each group of mice were used It can be clearly observed from figs. 1 and 2 that the to evaluate the model of femoral head osteonecrosis femoral head of the blank group had normal structure, in mice. HE staining results of femoral head tissue regular shape, regular bone trabecula arrangement. of each group of mice was observed, focusing on There was no abnormal signal in the femoral head, the structural changes of bone trabecula. Osteoblast and the hip joint was normal. In the model group, the growth, cell necrosis and apoptosis after activation morphology of the femoral head was changed. The of AMPK pathway in mouse model group was tested patchy irregular signal was seen in the femoral head. using an ELISA assay. The bone trabecula became thinner, the structure was The study was performed using SPSS 20.0 statistical loose and sparse, the subchondral bone was partially software. The data were expressed as mean±standard necrotic, and the bone marrow cavity was formed; the deviation and the data were verified with homogeneity structure of hip joint was normal. In the intervention test of variance. If p>0.05, the variance is equal, and group, the morphology of the femoral head was the comparison between the 2 means is analysed using significantly changed, the bone density decreased, the
152 Indian Journal of Pharmaceutical Sciences Special Issue 1, 2019 www.ijpsonline.com cavity of the bone marrow cavity increased significantly, clinical trials and animal experiments, cyclosporin A and the malunion of bone trabecula fracture occurred and PTFα can reduce bone mineral density and bone in the weight-bearing area, and the normal femoral mineralization. In this study, exogenous addition head tissue structure was lost. The difference between of cyclosporine A and PTFα to inhibit AMPK and the model group and the intervention group was P53-CyPD signalling pathways along dexamethasone statistically significant (p<0.05). administration can accelerate the apoptosis of mouse osteoblasts and accelerate the process of ONFH. It can be confirmed that dexamethasone, when used in Therefore, it can be concluded that AMPK and P53- large doses would destroy the bone structure, change CyPD signalling pathway play an important role in the the blood coagulation state, cause vascular disease and apoptosis of osteoblasts. bone tissue ischemia, and eventually lead to necrosis of the femoral head. Therefore, the mouse ONFH model The results of pathological examination are the can be successfully constructed by subcutaneous gold standard for the diagnosis of osteonecrosis. injection of dexamethasone. Both AMPK and The main pathological marker of dexamethasone- P53-CyPD signalling pathways are involved in the induced avascular necrosis of the femoral head is the regulation of osteoblast growth, development, and disappearance of bone cells in the lacuna. It can be apoptosis, and play an important role in responding seen from fig. 3 that in this study, the bone trabeculae to cytokine responses. In accordance with previous of the blank group were intact and arranged in an
a b c Fig. 1: Magnetic resonance images of the femoral head in mice (a) Blank group (b) model group (c) intervention group
a b c Fig. 2: Micro CT scans of the femoral head in mice (a) Blank group (b) model group (c) intervention group
a b c Fig. 3: Hematoxylin and eosin staining of ONFH in mice Hematoxylin and eosin staining representing osteonecrosis of femoral head (ONFH) in (a) Blank group (b) model group (c) intervention group of mice, 400X magnification Special Issue 1, 2019 Indian Journal of Pharmaceutical Sciences 153 www.ijpsonline.com orderly manner; the bone trabecula of the model group higher degree of apoptosis; while after the exogenous was significantly thinner and sparse, and some of the addition of AMPK activator AICAR, the apoptosis bone trabeculae were continuously damaged during was gradually improved. Thus, in the process of the fracture process. The bone trabecula structure combating apoptosis, the AMPK signalling pathway of the mice in the intervention group was obviously can be activated by growth factors and certain disordered, the cells were arranged irregularly, and the extracellular signals, regulating cell proliferation and dense cortex-like structure appeared, and the number differentiation. In this experiment, it was found that of bone cells in the lacuna decreased significantly. after the exogenous addition of AICAR, the AMPK The difference between the model group and the signalling pathway was activated, which can delay the intervention group was statistically significant (p<0.05). appearance of osteoblast aging, reduce inflammation, It can be concluded that dexamethasone had a certain inhibit vascular endothelial cell apoptosis, and improve destructive effect on the structure of bone trabecula and the bone density of the femoral head of mice and has a together with the inhibition of AMPK and P53-CyPD protective effect on the apoptosis of osteoblasts. signal transduction pathway can accelerate the process Ischemic osteonecrosis is a pathological process of bone tissue necrosis, and the mouse model of ONFH accompanied by a decrease in blood supply to was constructed in cooperation with dexamethasone. the bone, an increase in intra-osseous pressure, In this study, AMPK/P53-CyPD signalling pathway especially ONFH[7,8]. It is the result of a combination was activated by exogenously adding AMPK activator of traumatic and non-traumatic factors, and is the AICAR to mouse model tissue of dexamethasone- compensatory response of femoral head. With the induced ONFH (model group), and the role of AMPK/ collapse of osteonecrosis, it indicates the development P53-CyPD signalling pathway in inhibiting apoptosis and deterioration of the disease, and also determines of mouse osteoblasts was analysed. The study found that ONFH usually leads to osteonecrosis of the hip that after exogenous addition of AICAR, osteoblasts joint. Therefore, in the long run, total hip replacement grew slowly in the first 3 d. From the fourth day, the arthroplasty is an inevitable choice. According to growth rate of osteoblasts increased significantly, the survey, about 8 to 15 % of patients with ONFH suggesting that the cells entered the logarithmic in developed countries choose total hip replacement growth phase, of which on the 4th to 7th d, the trend arthroplasty. of logarithmic growth was observed. On the 8th d, the In recent years, with the changes in people's lifestyles growth of osteoblasts again became flat, suggesting and the widespread use of hormones, one of the serious that cell growth entered the plateau. The osteoblast consequences is the ischemic ONFH and its incidence growth curve is shown in fig. 4. is increasing year by year. High-dose, continuous use The absorbance after ELISA can indirectly indicate the of hormonal drugs may have an effect on bone tissue, apoptosis of osteoblasts. The higher the absorbance, which may lead to the apoptosis of osteoblasts, fat the higher the apoptotic rate of osteoblasts. The results cells, in the femoral head, thereby causing a series showed that the mice cells in the model group had a of pathological changes such as tissue ischemia. By regulating AMPK and P53-CyPD signalling pathway, 55 it has certain influence on hormone-induced osteoblast 5 apoptosis. In this paper, this was used as an entry point to further analyse the mechanism of activation 45 of AMPK/P53-CyPD signalling pathway to inhibit m
n apoptosis and promote cell formation.
4 7 5
A In this study, the commonly used hormone 35 dexamethasone was used to induce this condition. 3 When used in large doses and continuously, it will destroy the bone structure, change the blood 25 coagulation state, cause vascular disease and bone 2 4 6 8 1 tissue ischemia, and eventually lead to ONFH. By Culture time (d) exogenously adding cyclosporine A and PTFα to inhibit Fig. 4: Osteoblast growth curve AMPK and P53-CyPD signalling pathways based on 154 Indian Journal of Pharmaceutical Sciences Special Issue 1, 2019 www.ijpsonline.com the administration of dexamethasone, it can accelerate 4. Troncone M, Cargnelli SM, Villani LA, Isfahanian N, the apoptosis of mouse osteoblasts and accelerate the Broadfield LA, Zychla L, et al. Targeting metabolism and AMP-activated kinase with metformin to sensitize non-small process of ONFH. Apoptosis was gradually improved cell lung cancer (NSCLC) to cytotoxic therapy: translational by exogenously adding AMPK activator AICAR to biology and rationale for current clinical trials. Oncotarget dexamethasone-induced ONFH. Therefore, it was 2017;8(34):57733. concluded that AMPK and P53-CyPD signalling 5. de Almeida AJPO, Ribeiro TP, de Medeiros IA. Aging: pathway play an important role in osteoblast apoptosis Molecular Pathways and Implications on the Cardiovascular System. Oxid Med Cell Longev 2017;2017:7941563. induced by dexamethasone, which not only can inhibit 6. Mogavero A, Maiorana MV, Zanutto S, Varinelli L, Bozzi F, osteoblast apoptosis, delay the process of femoral head Belfiore A, et al. Metformin transiently inhibits colorectal osteonecrosis, but also promote osteoblast production, cancer cell proliferation as a result of either AMPK activation thereby can promote the recovery of ONFH. or increased ROS production. Sci Rep 2017;7(1):15992. 7. Kamiya N, Yamaguchi R, Aruwajoye O, Adapala NS, Kim HK. Acknowledgement: Development of a mouse model of ischemic osteonecrosis. Clin Orthop Relat Res 2015;473(4):1486-98. This work was supported by Jiangsu Science and 8. Kamiya N, Kuroyanagi G, Aruwajoye O, Kim HKW. IL6 Technology Office (Grant No: BE2016674). receptor blockade preserves articular cartilage and increases bone volume following ischemic osteonecrosis in immature REFERENCES mice. Osteoarthritis Cartilage 2019;27(2):326-35.
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